Sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci.
ABSTRACT: Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 438-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 19 enterococcal type strains (Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus seriolicida, Enterococcus solitarius, and Enterococcus sulfureus). Sequence analysis of the sodA(int) fragments enabled reliable identification of 18 enterococcal species, including E. casseliflavus-E. flavescens and E. gallinarum. The sodA(int) fragments of E. casseliflavus and E. flavescens were almost identical (99.5% sequence identity), which suggests that they should be associated in a single species. Our results confirm that the sodA gene constitutes a more discriminative target sequence than 16S rRNA gene in differentiating closely related bacterial species.
Project description:The 16S rRNA sequences of enterococcal species E. faecium, E. faecalis, E. gallinarum, E. casseliflavus/flavescens, E. dispar, E. pseudoavium, E. sulfureus, E. malodoratus, E. raffinosus, E. cecorum, E. hirae, E. saccharolyticus, E. seriolicida, E. mundtii, E. avium, E. durans, E. columbae, and E. solitarius are presented herein. These data were utilized to confirm the species identification of two nonmotile E. gallinarum isolates which had been previously phenotypically identified as E. faecium. The implications of this finding are discussed.
Project description:The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus durans, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, and Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus columbae, Enterococcus faecalis, Enterococcus sulfureus, Enterococcus saccharolyticus, and Enterococcus solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes.
Project description:Data from four recent studies (S. H. Goh et al., J. Clin. Microbiol. 36:2164-2166, 1998; S. H. Goh et al., J. Clin. Microbiol. 34:818-823, 1996; S. H. Goh et al., J. Clin. Microbiol. 35:3116-3121, 1997; A. Y. C. Kwok et al., Int. J. Syst. Bacteriol. 49:1181-1192, 1999) suggest that an approximately 600-bp region of the chaperonin 60 (Cpn60) gene, amplified by PCR with a single pair of degenerate primers, has utility as a potentially universal target for bacterial identification (ID). This Cpn60 gene ID method correctly identified isolates representative of numerous staphylococcal species and Streptococcus iniae, a human and animal pathogen. We report herein that this method enabled us to distinguish clearly between 17 Enterococcus species (Enterococcus asini, Enterococcus rattus, Enterococcus dispar, Enterococcus gallinarum, Enterococcus hirae, Enterococcus durans, Enterococcus cecorum, Enterococcus faecalis, Enterococcus mundtii, Enterococcus casseliflavus, Enterococcus faecium, Enterococcus malodoratus, Enterococcus raffinosus, Enterococcus avium, Enterococcus pseudoavium, Enterococcus new sp. strain Facklam, and Enterococcus saccharolyticus), and Vagococcus fluvialis, Lactococcus lactis, and Lactococcus garvieae. From 123 blind-tested samples, only two discrepancies were observed between the Facklam and Collins phenotyping method (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27:731-734, 1989) and the Cpn60 ID method. In each case, the discrepancies were resolved in favor of the Cpn60 ID method. The species distributions of the 123 blind-tested isolates were Enterococcus new sp. strain Facklam (ATCC 700913), 3; E. asini, 1; E. rattus, 4; E. dispar, 2; E. gallinarum, 20; E. hirae, 9; E. durans, 9; E. faecalis, 12; E. mundtii, 3; E. casseliflavus, 8; E. faecium, 25; E. malodoratus, 3; E. raffinosus, 8; E. avium, 4; E. pseudoavium, 1; an unknown Enterococcus clinical isolate, sp. strain R871; Vagococcus fluvialis, 4; Lactococcus garvieae, 3; Lactococcus lactis, 3; Leuconostoc sp., 1; and Pediococcus sp., 1. The Cpn60 gene ID method, coupled with reverse checkerboard hybridization, is an effective method for the identification of Enterococcus and related organisms.
Project description:The highly conserved central loop of domain V of 23S RNA (nucleotides 2042 to 2628; Escherichia coli numbering) is implicated in peptidyltransferase activity and represents one of the target sites for macrolide, lincosamide, and streptogramin B antibiotics. DNA encoding domain V (590 bp) of several species of Enterococcus was amplified by PCR. Twenty enterococcal isolates were tested, including Enterococcus faecium (six isolates), Enterococcus faecalis, Enterococcus avium, Enterococcus durans, Enterococcus gallinarum, Enterococcus casseliflavus (two isolates of each), and Enterococcus raffinosus, Enterococcus mundtii, Enterococcus malodoratus, and Enterococcus hirae (one isolate of each). For all isolates, species identification by biochemical testing was corroborated by 16S rRNA gene sequencing. The sequence of domain V of the 23S rRNA gene from E. faecium and E. faecalis differed from those of all other enterococci. The domain V sequences of E. durans and E. hirae were identical. This was also true for E. gallinarum and E. casseliflavus. E. avium differed from E. casseliflavus by 23 bases, from E. durans by 16 bases, and from E. malodoratus by 2 bases. E. avium differed from E. raffinosus by one base. Despite the fact that domain V is considered to be highly conserved, substantial differences were identified between several enterococcal species.
Project description:The VanC phenotype, as found in Enterococcus gallinarum, E. casseliflavus, and E. flavescens, is characterized by intrinsic low-level resistance to vancomycin. The nucleotide sequences of the vanC-1 gene in E. gallinarum, the vanC-2 gene in E. casseliflavus, and the vanC-3 gene in E. flavescens have been reported, although there is some disagreement as to whether E. flavescens is a legitimate enterococcal species. Previous attempts to differentiate the vanC-2 and vanC-3 genes by PCR analysis have been unsuccessful. The purpose of the present study was to detect and differentiate the three vanC determinants and examine the distribution of these genes in a collection of both typical and atypical enterococci. The 796-bp vanC-1 PCR product was amplified only from E. gallinarum isolates. As expected, due to the extensive homology in the vanC-2 and vanC-3 gene sequences, all of the E. casseliflavus and E. casseliflavus/flavescens isolates produced the 484-bp vanC-2 PCR product, although the E. gallinarum isolates were negative. Only the E. casseliflavus/flavescens isolates produced the 224-bp vanC-3 product. Using the three sets of primers, we were able to detect and distinguish the vanC-1, vanC-2, and vanC-3 genes from both typical and atypical enterococci strains. Antimicrobial susceptibility tests and analysis of genomic DNA by pulsed-field gel electrophoresis were also performed, but the results indicated that they were not able to distinguish among strains possessing the three vanC genotypes.
Project description:BACKGROUND:This study was aimed to investigate the intestinal microbiota in racing pigeons with regard to Enterococcus species distribution, virulence factors and antibiotic susceptibility. Three methods (API, Multiplex sodA-PCR, 16S rRNA sequencing) were compared for Enterococcus species identification. Cloacal samples from 179 apparently healthy pigeons of 13 different flocks were tested. RESULTS:Multiplex sodA-PCR and 16S rRNA gene sequencing showed almost perfect agreement in Enterococcus species identification. Isolates were identified as Enterococcus columbae (34.5%), Enterococcus hirae (20.7%), Enterococcus faecalis (11.7%), Enterococcus faecium (11.7%), Enterococcus gallinarum (9%), Enterococcus mundtii (4.8%), Enterococcus casseliflavus (3.4%), Enterococcus cecorum (2.1%), Enterococcus durans (2.1%). More Enterococcus species were found after the race season than before. The study showed differences between Enterococcus species in relation to 68.8% (22/32) biochemical parameters. Six out of seven virulence genes were detected: gelE (43.5%), asa1 (42.1%), efaA (30.3%), ace (30.3%), cylA (27.6%), and esp (9%). None of the isolates harboured hyl gene. Overall 15.2% of Enterococcus isolates produced gelatinase, but 66.7% gelE genes were silent. Enterococcus faecalis showed the most often efaA, ace and gelatinase activity than other enterococcal species. Nearly all isolates (93.1%) were resistant to at least one antibiotic. The most frequent resistance was to enrofloxacin (80%), doxycycline with teicoplanin (73.1%), erythromycin (49.7%). The study revealed significant differences between some enterococcal species in the antibiotic susceptibility to different antibiotics. Enterococcus columbae and E. cecorum showed significantly more frequent resistance to chloramphenicol than other enterococci. The presence of VRE (19.3%), HLGR (2.8%) and no LRE were found. Overall 30.3% of isolates were positive for vancomycin resistance genes, where vanC1 (E. gallinarum), vanC2-C3 (E. hirae, E. casseliflavus), vanB (E. columbae) predominated. CONCLUSIONS:We conclude, that intestinal microbiota in racing pigeons is composed by 9 different Enterococcus species. Given that racing pigeons are kept in close contact with humans and backyard animals, combined with their long-distance flight abilities, they can serve as potential source of virulent and antibiotic resistant Enterococcus spp. in the environment.
Project description:This study aimed to evaluate the clinical outcomes of vancomycin-resistant enterococcal bloodstream infections (VRE BSI) caused by Enterococcus gallinarum or Enterococcus casseliflavus. Variables associated with treatment failure were determined and treatment options were compared. This was a national retrospective study of hospitalised Veterans Affairs patients with non-faecium, non-faecalis VRE BSI. The primary outcome was treatment failure, defined as a composite of: (i) 30-day all-cause mortality; (ii) microbiological failure; and (iii) 30-day VRE BSI recurrence. Stepwise Poisson regression was conducted to determine variables associated with treatment failure. In total, 48 patients were included, with 29 cases (60.4%) caused by E. gallinarum and 19 cases (39.6%) caused by E. casseliflavus. Among these cases, 20 (41.7%) were treated with an anti-VRE agent (linezolid or daptomycin) and 28 (58.3%) were treated with an anti-enterococcal ?-lactam. Overall, 30-day mortality was 10.4% (5/48) and composite treatment failure was 39.6% (19/48). In multivariate analysis, treatment with an anti-enterococcal ?-lactam was associated with increased treatment failure in comparison with anti-VRE therapy (adjusted risk ratio = 1.73, 95% confidence interval 1.06-4.97; P = 0.031). Overall, treatment with linezolid or daptomycin for vancomycin-resistant E. gallinarum or E. casseliflavus BSI resulted in improved clinical outcomes in comparison with anti-enterococcal ?-lactam treatment.
Project description:Using degenerate oligonucleotides complementary to sequences encoding conserved amino acid motifs in D-alanine-D-alanine (Ddl) ligases, we have amplified ca. 600-bp fragments from Enterococcus casseliflavus ATCC 25788 and Enterococcus flavescens CCM439. Sequence analysis of the amplification products indicated that each strain possessed two genes, ddlE. cass. and vanC-2, and ddlE. flav. and vanC-3, respectively, encoding Ddl-related enzymes. The fragments internal to the vanC genes were 98.3% identical. The vanC-2 gene was cloned into Escherichia coli and sequenced. Extensive similarity (66% nucleotide identity) was detected between this gene and vanC-1 from Enterococcus gallinarum (S. Dutka-Malen, C. Molinas, M. Arthur, and P. Courvalin, Gene 112:53-58, 1992), suggesting that the vanC genes are required for intrinsic low-level resistance to vancomycin. The partial deduced amino acid sequences of ddlE. cass. and ddlE. flav. were identical and closely related to that of the Ddl ligase of Enterococcus faecalis (79% identity). In Southern hybridization experiments, only DNA from E. casseliflavus and E. flavescens hybridized to probes internal to the vanC-2 and ddlE. cass. genes.
Project description:BACKGROUND:Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted. RESULTS:VRE isolates, including E. faecalis (n?=?24), E. faecium (n?=?11), E. casseliflavus (n?=?2) and E. gallinarum (n?=?2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium (p?<?0.001) and E. faecalis (p?<?0.001) and with the number of AMR genes in E. faecium (p?=?0.005). Genes conferring vancomycin resistance, including vanA and vanM (E. faecium), vanG (E. faecalis), and vanC (E. casseliflavus/E. gallinarum), were detected in 20 genomes. The most prominent functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium, E. faecalis, E. casseliflavus and E. gallinarum, respectively. Virulence genes were more common in E. faecalis and E. faecium, than E. casseliflavus and E. gallinarum. A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium. Phylogenetic analysis demonstrated differential clustering of isolates based on original source but not WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers. CONCLUSIONS:There was no discernible difference between enterococcal genomes from the CAS and BAF WWTPs. E. faecalis and E. faecium have smaller genomes and harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp.
Project description:The enterococci are low-GC Gram-positive bacteria that have emerged as leading causes of hospital-acquired infection. They are also commensals of the gastrointestinal tract of healthy humans and most other animals with gastrointestinal flora and are important for food fermentations. Here we report the availability of draft genome sequences for 28 enterococcal strains of diverse origin, including the species Enterococcus faecalis, E. faecium, E. casseliflavus, and E. gallinarum.